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PubMed1988

Postirradiation alterations of neuronal chromatin structure.

Previous work from our laboratory suggested that neuronal chromatin structure may be altered immediately after exposure to ionizing radiation. In the present study, whole brains of 4-month-old male Fisher 344 rats were irradiated with a dose of 25 Gy. The kinetics of restoring the chromatin structure to its unirradiated state was investigated in rat cerebellar neurons using three different approaches: (1) measurement of changes in the DNA superhelical structure by the fluorescent halo assay, (2) measurement of changes in chromatin accessibility to digestion by micrococcal nuclease, and (3) measurement of changes in the accessibility of the nuclear-matrix-associated DNA to digestion by DNase I. Immediately after irradiation, the topological constraints on the DNA loops were altered, the chromatin was more accessible to m. nuclease digestion, and the DNA associated with the nuclear matrix was more resistant to digestion by DNase I. Return of the chromatin structure to its unirradiated state as measured by each of the three methods followed biphasic kinetics with the fast phase having a half-time of several minutes and the slow phase having a half-time of several hours. The kinetics are similar to that previously reported for repair of radiation-induced DNA damage in mammalian cells. Although the independent assays used in this study seemed to follow the same kinetics, their relationship at the molecular level remains to be determined.

Animals↗
PubMed2004

[The potential role of nuclear matrix attachment regions (MARs) in regulation of gene expression].

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However, it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Because integration seemed to occur randomly in the plant genome, it was thought that some transgenes would be integrated in a relatively uncondensed, transcriptionally active chromatin environment, while others in a condensed, transcriptionally inert chromatin structure. Nuclear matrix attachment regions (MARs) are defined as DNA sequences that bind preferentially to the proteins of the nuclear matrix. They typically are localized at the borders of gene domains, implicating them in the formation of individual loops of higher order chromatin structure and transcription regulation. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable espression in transgenic plants, most likely by minimizing gene silencing. In this review, we focus mainly on novel findings and our observations concerning the function of MARs in transcription regulation. Our objective is not only to summarize the current data and present several possible models to explain MAR effects on the transcription regulation, but also to point out some open questions involving the utilization of MARs in constructing high efficient expression vectors.

Chromatin↗
PubMed2001

DNA fragmentation in mammalian cells exposed to various light ions.

Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/micrometer protons, 123 keV/micrometer helium-4 ions and gamma rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respect to that induced by comparable doses of gamma rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for gamma rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage repairability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by gamma rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

Animals↗
PubMed2026

Twisting the End Game: How Telomere Chromatin Modifications Shape Telomere Maintenance.

Cell division inevitably shortens telomeric DNA owing to the end-replication problem. Eukaryotic chromosomes possess specialized telomere structures to maintain genomic stability. In most proliferative cells, telomerase adds telomeric repeats during S-phase. In differentiated cells where telomerase is silenced, telomeres shorten progressively, thereby compromising genomic integrity. Consequently, cancer cells universally activate alternative telomere maintenance mechanisms during malignant transformation: ~80% reactivate telomerase, while a portion of the rest rely on BIR (break-induced replication)-mediated homologous recombination-based ALT (alternative lengthening of telomeres). Although these mechanisms are stable once established, the initial determinants influencing a cancer cell's choice remain poorly understood. This review discusses recent molecular insights into how telomeric chromatin properties profoundly impact this choice. After briefly introducing telomere chromatin characteristics and key players in its maintenance and dynamics, we discuss the mechanisms by which cancer cells acquire distinct telomere replication capabilities. In particular, we present an in-depth analysis linking telomere heterochromatin status to ALT. Furthermore, based on recent advances, we propose a coupled feedforward loop model explaining how the ALT state becomes "locked in" once initiated. Finally, we offer novel perspectives on rational, telomere-centric therapeutic interventions for ALT-positive cancers, focusing on strategies designed to disrupt such feedforward loops by manipulating telomeric chromatin structure.

Humans↗
PubMed1989

Anti-topoisomerase II recognizes meiotic chromosome cores.

At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II - a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation - is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.

Animals↗
PubMed2005

3D analysis of chromosome architecture: advantages and limitations with SEM.

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the "dynamic matrix model" for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.

Chromosomes, Plant↗
PubMed1992

Isolation of a novel nuclear glycoprotein from pig kidney.

A nuclear glycoprotein with an apparent Mr of 66,000 Da has been isolated from pig kidney chromatin after extraction with urea, guanidine-HCl and 2 M NaCl, and some of its structural features have been characterized. It belongs to the group of N-glycosylated proteins, which in the nucleus has so far received little attention. From its monosaccharide composition and recognition by lectins its oligosaccharides appear to be of high mannose and/or hybrid types. Some properties of its protein moiety suggest that it has a role in the packing of the DNA loops in the condensed chromatin.

Amino Acid Sequence↗
PubMed2004

High-affinity binding of tumor-suppressor protein p53 and HMGB1 to hemicatenated DNA loops.

We have recently observed that chromatin architectural protein HMGB1 (previously reported to be involved in numerous biological processes such as DNA replication, recombination, repair, tumor growth, and metastasis) could bind with extremely high affinity (K(d) < 1 pM) to a novel DNA structure that forms a DNA loop maintained at its base by a hemicatenane (hcDNA). The loop of hcDNA contains a track of repetitive sequences derived from CA-microsatellites. Here, we report using a gel-retardation assay that tumor-suppressor protein p53 can also bind to hcDNA. p53 is a crucial molecule protecting cells from malignant transformation by regulating cell-cycle progression, apoptosis, and DNA repair by activation or repression of transcription of its target genes by binding to specific p53 DNA-binding sites and/or certain types of DNA lesions or alternative DNA structures. The affinity of p53 for hcDNA (containing sequences with no resemblance to the p53 DNA consensus sequence) is >40-fold higher (K(d) approximately 0.5 nM) than that for its natural specific binding sites within its target genes (Mdm2 promoter). Binding of p53 to hcDNA remains detectable in the presence of up to approximately 4 orders of magnitude of mass excess of competitor linear DNA, suggesting a high specificity of the interaction. p53 displays a higher affinity for hcDNA than for DNA minicircles (lacking functional p53-specific binding sequence) with a size similar to that of the loop within the hcDNA, indicating that the extreme affinity of p53 for hcDNA is likely due to the binding of the protein to the hemicatenane. Although binding of p53 to hcDNA occurs in the absence of the nonspecific DNA-binding extreme carboxy-terminal regulatory domain (30-C, residues 363-393), the isolated 30-C domain (but not the sequence-specific p53 "core domain", residues 94-312) can also bind hcDNA. Only the full-length p53 can form stable ternary complexes with hcDNA and HMGB1. The possible biological relevance of p53 and HMGB1 binding to hemicatenanes is discussed.

Animals↗
PubMed2000

Rearrangement of chromatin domains in cancer and development.

Both the accomplishment of developmental programs and neoplastic transformation are linked to changes in the long-range organization of chromatin, in particular, DNA loop domains. The development of new methods that allow the study of interactions between the bases of DNA loops and the proteins of the nuclear matrix will help our understanding of the molecular mechanisms in such changes. These methods should also allow the establishment of a fingerprint "signature" for many cancers that may serve for diagnostic purposes. J. Cell. Biochem. Suppl. 35:54-60, 2000.

Animals↗
PubMed1994

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy.

HeLa metaphase chromosomes were examined by means of "in lens" field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Chromosomes, Human↗
PubMed2005

Host cell targets of immediate-early protein BICP22 of bovine herpesvirus 1.

The immediate-early (IE) protein BICP22 of bovine herpesvirus 1 (BHV-1) acts as transrepressor protein on viral promoters of different kinetic classes. In the present work, we looked for host cell targets of BICP22 using a yeast two-hybrid system and identified seven candidates: (1) JIK, a serine/threonine kinase of the sterile 20 protein (STE20) family that inhibits stress-related pathways; (2) cAMP response element binding protein-like 2 (CREBL2), which in its bZip domain shares homology with CREB, modulating transcription of cAMP responsive genes; (3) DNA-dependent ATPase and helicase (ATRX), a protein of the SNF2 family altering nucleosome structure; (4) scaffold attachment factor B (SAF-B), which helps to organize chromatin into topologically separated loops; (5) peptidylglycine alpha-amidating monooxygenase COOH-terminal interactor protein 1 (PAMCIP1), involved in regulation of the secretory pathway in the perinuclear area; (6) zinc finger protein (ZNF38) found in proliferating cells and possibly associated with meiosis in male and female gametogenesis; (7) FLJ22709, hypothetical protein conserved among various species, containing an occludin/ELL domain. To confirm some of the interactions by confocal fluorescence microscopy, BICP22 was tagged with red fluorescent protein in an amplicon, and selected target sequences were tagged with green fluorescent protein in plasmid expression vectors. Upon amplicon transduction of Vero cells and plasmid transfection, CREBL2 and ZNF38 both colocalized with BICP22 in distinct nuclear domains.

Amino Acid Sequence↗
PubMed1999

Poly(ADP-ribose) polymerase and Ku autoantigen form a complex and synergistically bind to matrix attachment sequences.

Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain. Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo. By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription. In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates. We further demonstrate that PARP and Ku autoantigen form a molecular complex in vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced. ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex. These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function.

Antigens, Nuclear↗
PubMed2004

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. II. Simulation with a generalized broken stick model.

PURPOSE: To characterize the differences among the experimental DNA fragmentation spectra induced in Chinese hamster V79 cells by gamma-rays, low-energy protons and alpha-particles through the use of a phenomenological model. MATERIALS AND METHODS: A model of DNA fragmentation was developed as a generalization of the broken-stick model, in which the double-strand breaks induced by radiation were considered randomly placed, but in which the manifestly non-random fragmentation of the control sample was fully taken into account and considered as the initial fragment distribution. Further, an analytical method was introduced that allowed an evaluation of the deviation from randomness of the fragmentation induced by radiation. RESULTS: The analysis of the experimental distribution of DNA fragments showed that there was a progressive departure from randomness in radiation-induced fragmentation going from gamma-rays to protons and then to alpha-particles. This deviation was characterized by an enhanced induction of fragments, and therefore by a larger correlation of double-strand breaks, in the experimental range of lower molecular weights. CONCLUSION: The analysis shows that low-energy light ions induce DNA fragmentation, at the loop level of the chromatin organization, that can be significantly non-random. The same analysis can readily be applied at different length scales, and thus it could offer a basis for the study of the link between DNA damage, correlated at various spatial scales and biological end-points.

Alpha Particles↗
PubMed1991

DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells.

Mammalian sperm DNA is the most tightly compacted eukaryotic DNA, being at least sixfold more highly condensed than the DNA in mitotic chromosomes. To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin. This differs markedly from the bulkier DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. The overall organization of mammalian sperm DNA, however, resembles that of somatic cells in that both the linear arrays of sperm chromatin and the 30-nm solenoid filaments of somatic cell chromatin are organized into loop domains attached at their bases to a nuclear matrix. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored when the nuclear matrix is disrupted during decondensation. In somatic cells, proper function of DNA is dependent upon the structural organization of the DNA by the nuclear matrix, and the structural organization of sperm DNA is likely to be just as vital to the proper functioning of the spermatozoa.

Animals↗
PubMed1992

Biological significance of unwinding capability of nuclear matrix-associating DNAs.

Matrix attachment regions (MARs) are thought to separate chromatin into topologically constrained loop domains. A MAR located 5' of the human beta-interferon gene becomes stably base-unpaired under superhelical strain, as do the MARs flanking the immunoglobulin heavy chain gene enhancer; in both cases a nucleation site exists for DNA unwinding. Concatemerized oligonucleotides containing the unwinding nucleation site exhibited a strong affinity for the nuclear scaffold and augmented SV40 promoter activity in stable transformants. Mutated concatemerized oligonucleotides resisted unwinding, showed weak affinity for the nuclear scaffold, and did not enhance promoter activity. These results suggest that the DNA feature capable of relieving superhelical strain is important for MAR functions.

Base Sequence↗
PubMed1997

Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2.

Here, we describe the cloning and further characterization of chicken ARBP, an abundant nuclear protein with a high affinity for MAR/SARs. Surprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spanning 125 amino acids in the N-terminal halves is 96.8% identical between chicken ARBP and rat MeCP2. A deletion mutation analysis using Southwestern and band shift assays identified this highly conserved region as the MAR DNA binding domain. Alignment of chicken ARBP with rat and human MeCP2 proteins revealed six trinucleotide amplifications generating up to 34-fold repetitions of a single amino acid. Because MeCP2 was previously localized to pericentromeric heterochromatin in mouse chromosomes, we analyzed the in vitro binding of ARBP to various repetitive sequences. In band shift experiments, ARBP binds to two chicken repetitive sequences as well as to mouse satellite DNA with high affinity similar to that of its binding to chicken lysozyme MAR fragments. In mouse satellite DNA, use of several footprinting techniques characterized two high-affinity binding sites, whose sequences are related to the ARBP binding site consensus in the chicken lysozyme MAR (5'-GGTGT-3'). Band shift experiments indicated that methylation increased in vitro binding of ARBP to mouse satellite DNA two- to fivefold. Our results suggest that ARBP/MeCP2 is a multifunctional protein with roles in loop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.

Amino Acid Sequence↗
PubMed2004

Circadian and light-induced transcription of clock gene Per1 depends on histone acetylation and deacetylation.

Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. Here, we show that the state of acetylated histones fluctuated in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) repressed transcription with HDACs and mSin3B, which was relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induced endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which interacted with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulated rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. We also showed that TSA administration into the lateral ventricle induced mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation.

Acetylation↗
PubMed1997

Scaffold attachments within the human genome.

It is generally agreed that, above the level of the 30 nm fibre, eukaryotic chromatin is constrained into loops, but there is disagreement about the nature of the substructure that serves to anchor loops and the DNA sequences that act as the attachment sites. This problem may stem from the very different methods that all purport to separate loop and attached DNAs. We have tested ideas about how the genome is arranged into loops by analysing the average loop size over different cytologically resolvable regions of human chromosomes using fluorescence in situ hybridisation with loop and attached DNA fractions. Variations in average loop size, along and between chromosomes, measurable at this level of resolution were small but significant and were dependent on the extraction method. This emphasises the fundamental differences between the nuclear substructure probed by different protocols. DNA attached to the nuclear 'scaffold' or 'matrix' hybridises preferentially to gene-poor regions of the genome (G-bands). Conversely, fractions attached to the nuclear 'skeleton' hybridise preferentially to gene-rich R-bands and sites of high levels of transcription. The inactive X chromosome has a deficit of associations with the nuclear skeleton but not with the matrix or scaffold. A large excess of attached sequences is found at some sites of constitutive heterochromatin, but not at centromeres.

Cell Nucleus↗